Conventional analysis of small samples may entail laborious and time-consuming analysis under a microscope. More efficient analysis of samples may be performed using automated microscopy technology.
For example, common testing protocol is FISH. Typically FISH analysis is done by manual inspection of tissue samples by a skilled microscopist. In addition to correctly identifying the dot and its color, other size and shape characteristics must be categorized to correctly identify the condition. The analysis is made more difficult by the time constraints imposed by the phenomena. The microscopist, therefore, must be exhaustively trained to perform the examination. Even under the best conditions, the process has been proven to be tedious, lengthily and subject to human error.
The application of automated microscopy has the potential to overcome many of the shortcomings of the manual approach. The automatic microscope can reliably identify the fluorescent dots in a tissue sample, accurately determine their color, categorize them based on shape and size, and perform the summary analysis necessary to determine the presence or absence of the targeted condition without the inevitable subjective factors introduced by a human operator all in a timely manner.
Some widely-used Down syndrome test employ FISH analysis. For example, the AneuVysion test uses colored DNA probes that attach to specific chromosomes in the amniotic fluid cells. Unlike karyotyping, this test doesn't require growth of the cells in a laboratory incubator. Thus, cells can be analyzed within hours after the DNA probes are added. After the DNA probes are mixed with the cells, they attach to specific chromosomes. Different probes carry different colored fluorophores to allow for differentiation. For example, when a probe finds chromosome 21, it attaches in a very specific spot, and, for example, an orange-colored signal within the fetal cell is visible through the microscope. A laboratory technologist then must manually count the number of colored spots in each cell and determine if there are the two number 21 chromosomes as expected or if there are three 21 chromosomes indicating Down syndrome. The same procedure may be performed simultaneously for chromosomes 13, 18, X and Y with each chromosome appearing as a different colored spot. Thus if three spots for chromosome 13 or 18 are observed, then the fetus has trisomy 13 or trisomy 18. Turner or Klinefelter syndromes are detected by the probes for the X and Y chromosomes.
In the case of FISH analysis, the viewer must be able to discriminate and quantify the various emanations coming from the fluorphores. The difficulty in doing this may be said to be greatly increased when an automated microscope is utilized. An automated microscope must be capable of quantifying dim emanations of various wavelength fluorescent light from linked dye markers associated with tissue sample. When extremely rare cells are being analyzed, such as, for example without limitation, when fetal cells are being analyzed, there needs to be the ability for the microscope to detect signals from among large quantities of cells etc.
When rare cells or elements are to be detected, there may be the need to examine many slides to make a diagnosis or determination pertaining to the subject, material etc. from which the analyzed samples were taken. For example with respect to rare cells such as fetal cells in a maternal blood sample, this may require the examination of over 200 slides for appropriate determination. The sample on each of these slides, may in turn, cover an area corresponding to a large number of microscopic fields of view. Thus many microscopic fields of view may be needed to be examined to perform a single analysis. Of course, it would be desirable to provide a single image capture time as low as possible, for example, in some cases of approximately 5 milliseconds or less.
In FISH analysis, examination may be made more difficult by a low level of emitted fluorescent light that may be produced, low half life and a relatively short emission period. A further challenge that may be faced is that the markers may fluoresce at differing wavelengths which serve as identification parameters.
The practical implementation of an automated microscope suitable for the detection of rare components in a sample, such as rare cells in a tissue sample, may require image capture technology that satisfies several diverse requirements, for example, include high sensitivity, fast capture speed, high frame rate, and superior wavelength resolution.